In the mammalian central nervous system, L-glutamate serves as a major excitatory neurotransmitter. The interaction of glutamate with its membrane-bound receptors is believed to play a role in many important neuronal processes, including, for example, fast synaptic transmission, synaptic plasticity and long-term potentiation. These processes are fundamental to the maintenance of life and normal human abilities such as learning and memory. Monaghan D. T. et al., 8 Neuron 267 (1992).
Pharmacological characterization of receptors for L-glutamate has led to their classification into two families based on their biological function: the ionotropic receptors which are directly coupled to cation channels in the cell membrane, and the metabotropic receptors which function through coupling to G-proteins. A number of ionotropic receptors have been further characterized on the basis of the relatively specific agonists by which they can be activated. One major group comprises those receptors activated by N-methyl-D-aspartate (NMDA), which appears to have multiple allosteric modulatory sites. The other two groups consist of those receptors activated by kainate and/or amino-3-hydroxy-5-methyl-4-isoxozole propionate (AMPA). Collingridge G. L. et al., 40 Pharmacol. Rev. 143 (1989).
Molecular cloning studies of rodent ionotropic receptors have recently provided some information on the molecular structure of these proteins. The cDNAs for seven different subtypes of the kainate/AMPA group have been characterized. Heinemann S. et al., PCT publication, WO91/06648 (1991), Keinanen K. et al., 249 Science 556 (1990), Sakimura K. et al., 272 FEBS Lett. 73 (1990), Werner P. et al., 341 Nature 742 (1991), Bettler B. et al., 8 Neuron 257 (1992). Splice variants, referred to as "flip" and "flop", of same of these have been characterized as well. Sommer B. et al., 249 Science 1580 (1990). In addition, two members of the NMDA group have been cloned. Moriyoshi, K. et al., 354 Nature 31 (1991) and Meguro H. et al., 357 Nature 70 (1992). An NMDA-related protein has also been reported. Kumar K. N. et al., 354 Nature 70 (1991). These proteins share varying degrees of homology with one another and are therefore believed to represent a gene superfamily. Based on analogy with other better characterized ion channel receptors, glutamate ionotropic receptors are expected to exist in vivo within the cell membrane as heteromeric multisubunit assemblies of these subunits. Unwin N., 3 Neuron 665 (1989).
Moreover, at least two human glutamate receptors have been reported as cloned. Puckett C. et al., 88 Proc. Nat. Acad. Sci. 7557 (1991) and Sun W. et al., 89 Proc. Nat. Acad. Sci. 1443 (1992). The glutamate receptor cloned by Puckett et al. was named GluHI and was later identified to be the "flip" version of this particular receptor. The Sun W. et al. reference refers to the glutamate receptor they cloned as the HBGR1 receptor and explains that HBGR1 is presumed the "flop" version of GluHI. Sun et al. also discloses the possible existence of, but does not describe in detail, a partial clone of HBGR2, or human GluR2.
In addition to its role in normal human physiology, interaction of L-glutamate with its receptors is believed to play a key role in many neurological disorders such as stroke, epilepsy and head trauma, as well as neurodegenerative processes such as Alzheimer's disease. Olney R. W., 17 Drug Dev. Res., 299 (1989). For this reason, understanding the molecular structure of human L-glutamate receptors will be important for understanding these disease processes as well as for furthering the search for effective therapeutic agents. Up to the present, the search for therapeutic agents which will selectively bind and modulate the function of human glutamate receptors has been hampered by the unavailability of homogeneous sources of receptors to use for screens and tests of potential drug candidate compounds. The brain tissues commonly used by pharmacologists presently are derived from experimental animals (non-human) and furthermore contain mixtures of various types of glutamate receptors.
In searching for drugs for human therapy it is desirable to use receptors that are more analogous to those in the intact human brain than are the rodent receptors employed to date. The current invention provides a human receptor and functional equivalents thereof which can be used to search for drugs which modulate this receptor.
For purposes of clarity and as an aid in understanding the invention, as disclosed and claimed herein, the following items are defined below.
"Functional HSG1uR2"--A compound comprising SEQ ID NO:1 which, when alone or combined with another glutamate receptor, is capable of generating ion flow, binding glutamate, interacting with glutaminergic ligand, or performing in a manner consistent with a glutamate receptor.
"GluR2 receptor"--The amino acid sequence commonly associated with the rat ionotropic glutamate receptor 2.
"GluR3 receptor"--The amino acid sequence commonly associated with the rat ionotropic glutamate receptor 3.
"HSG1uR1 receptor"--The amino acid compound disclosed in copending application (attorney docket No. 8342, having inventors Burnett J. P., Mayne N. G., Sharp R. L. and Snyder Y. M.) or functional equivalents thereof.
"HSG1uR2 receptor"--The compound having amino acid sequence SEQ ID NO:1 or functional equivalents thereof.
"mRNA"--RNA which has been transcribed either in vivo or in vitro, including, for example, RNA transcripts prepared in vitro via transcription of coding sequences of DNA by RNA polymerase.
"Part of SEQ ID NO:1"--A sequence containing at least 6 consecutive amino acid residues or more and that corresponds to a sequence contained in SEQ ID NO:1.
"Physically detectable"--Any information which has been presented in humanly recognizable form, with or without the aid of intervening interpretation. For example, electrophysiological, chemical or biochemical data is considered within the realm of physically detectable information.
"Primer"--A nucleic acid fragment or its reverse complement which functions as template for enzymatic or synthetic elongation.
"Probe"--A nucleic acid compound or a fragment thereof, or their reverse complement, either of which is used to hybridize to other nucleic acids.
"SEQ ID NO:1 and functional equivalents thereof"--SEQ ID NO:1 and conservative alterations of the amino acid sequence of SEQ ID NO:1, wherein the conservative alterations result in a compound which exhibits substantially the same biological, biochemical, physical and structural qualities of SEQ ID:1.
"SEQ ID NO:2"--a DNA sequence which encodes SEQ ID NO:1.
"SEQ ID NO:3"--The DNA sequence ATGCAAAAGA TTATGCATAT TTCTGTCCTC CTTTCTCCTG TTTTATGGGG ACTGATTTT. This segment includes bases 1 through 60 of SEQ ID NO:2, counting from the 5' end.
"SEQ ID NO:4"--The DNA sequence GTAGG GATGG TTCAGTTTTC CACTTCGGAG TTCAGACTGA CACCCCACAT CGACAATTTG. This segment includes bases 136 through 195 of SEQ ID NO:2, counting from the 5' end.
"SEQ ID NO:5"--The DNA sequence AATTTTGCAA CTTATAAGGA AGGTTACAAC GTATATGGCA TCGAAAGTGT TAAAATTTAA. This segment includes bases 2593 through 2649 of SEQ ID NO:2, with a TAA stop codon added at the 3' end.
"Transfection"--Any transfer of nucleic acid into a host cell, with or without integration of said nucleic acid into genome of said host cell.